functional characterization of keap1 tcga mutants in ...matt walker feng yan alex rabinowitz hayes...
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Functional characterization of KEAP1 mutations in lung squamous cell carincoma Bridgid Hast Major Lab UNC Chapel Hill
KEAP1/NRF2 regulates intracellular redox homeostasis
CUL3
Risk
for D
isea
se
KEAP1 null
NRF2 null
Effective Prevention
NRF2 mediated transcription
Neurodegeration Cancer
NRF2 activity modulates survival via redox homeostasis
NRF2 target genes -Heme oxygenase 1 (HMOX1) -Glutathione synthesis (GCS) -NADH quinone oxidoreductase 1 (NQO1) -Multidrug resistance proteins (MRP)
*Mitigate acute spikes in ROS *Chemotherapeutic/xenobiotic clearance *Control metabolically-derived ROS
Pathway mutations in KEAP1/NRF2 signaling occur in squamous cell lung carcinoma
-178 total squamous cell lung carcinomas analyzed -Mutations in KEAP1 and NRF2 are mutually exclusive -Primarily in classical subtype -Collectively KEAP1, NRF2, and CUL3 mutations are altered in 34% of total samples The Cancer Genome Atlas Research Network, Nature 2012
KEAP1 mutations exhibit differential suppression of NRF2-mediated transcription
0
0.2
0.4
0.6
0.8
1
1.2
GFP
KE
AP1
R
554Q
R
320Q
W
554C
R
470C
G
423V
N
469f
s G
480W
D
422N
G
333C
L2
31V
P3
18L
G18
6R
S243
C
P318
de
l V
167F
R
71K
V15
5F
Fire
fly/R
en
illa
KEAP1 mutants differentially bind to interacting proteins
KEAP1 mutants differentially bind to interacting proteins
KEAP1 mutants differentially bind to interacting proteins
The KEAP1 mutants cluster into four classes
IVR 180-314
KELCH 315-359
KELCH 361-410
KELCH 412-457
KELCH 459-504
KELCH 506-551
KELCH 553-598
NTR 1-60
CTR 599-624
BTB 61-179
Class I: Strong binders of NRF2 but cannot suppress NRF2-mediated transcription
IVR 180-314
KELCH 315-359
KELCH 361-410
KELCH 412-457
KELCH 459-504
KELCH 506-551
KELCH 553-598
NTR 1-60
CTR 599-624
BTB 61-179
IVR 180-314
KELCH 315-359
KELCH 361-410
KELCH 412-457
KELCH 459-504
KELCH 506-551
KELCH 553-598
NTR 1-60
CTR 599-624
BTB 61-179
IVR 180-314
KELCH 315-359
KELCH 361-410
KELCH 412-457
KELCH 459-504
KELCH 506-551
KELCH 553-598
NTR 1-60
CTR 599-624
BTB 61-179
Class II: Do not bind NRF2 and cannot suppress NRF2
Class III: Weakly bind NRF2 and cannot suppress NRF2
Class IV: Behave like wildtype
R320Q D422N G423V
R470C G186R S243L V155F
W544C R554Q N469fs
G333C P318L G480W
V167F
L231V S224Y P318del R71K
KEAP1 mutants differentially bind to interacting proteins
-“Superbinders” only bind more NRF2
- Cannot suppress NRF2-mediated transcription
- Exhibit increased NRF2 half-life
- Have enhanced cell viability in response to chemotherapeutic insult Mechanism?
0
0.2
0.4
0.6
0.8
1
1.2
GFP
KE
AP1
R
554Q
R
320Q
W
554C
R
470C
G
423V
N
469f
s G
480W
D
422N
G
333C
L2
31V
P3
18L
G18
6R
S243
C
P318
de
l V
167F
R
71K
V15
5F
Fire
fly/R
en
illa
“Superbinders”: slow cyclers or subpar structures?
IVR
KELCH
NRF2
90°
D422 R470
D422 R470
P318 R320
NRF2 DLG and ETGE
-Class I mutants are on “bottom” of KELCH domain, not at KELCH/NRF2 interface -Mutants in IVR and linker between IVR and KELCH are also in Class I. Predicted to be near IVR/BTB interface -More likely that Class I mutants perturb KEAP1 structure than act as “superbinders” 0
0.2
0.4
0.6
0.8
1
1.2
GFP
KEA
P1
R55
4Q
R32
0Q
W55
4C
R47
0C
G42
3V
N46
9fs
G48
0W
D42
2N
G33
3C
L231
V
P318
L
G18
6R
S243
C
P318
de
l
V16
7F
R71
K
V15
5F
Fire
fly/R
en
illa
KEAP1 cysteine residues are stress-specific
-C151 forms adducts with electrophiles -H129, K131, R135, K150, and H154 comprise microenvironment that alters reactivity of C151
-H225/C226 and C613 are reactive to heavy metals -C288 specific reactivity to alkenals
McMahon et al, PNAS 2010
Is cysteine reactivity in KEAP1 altered in cancer?
KEAP1 mutations cluster
Are clustered mutations “pointing” to important regions of KEAP1?
Cys 241, 249, 319, 368, 434, 489 have been shown to react with electrophilic fatty acids as well as sulforaphane
240 320-350 430 Approximate Residue
KEAP1 mutations are hypomorphic and can be further inactivated by interacting proteins
(DPP3)
-Overexpression of the ETGE-containing protein DPP3 further activates NRF2 signaling in a KEAP1 mutant background -DPP3 is overexpressed in tumor verses normal lung squamous cell carcinoma (p=4.6e-14)
Summary
-Mutations in KEAP1 from lung squamous cell carcinoma can be grouped into four phenotypic classes -The “superbinder” class exhibits enhanced NRF2 activity and stability, and is likely a result of structural changes in the KEAP1 homodimer -KEAP1 mutations in cancer cluster around cysteines with reactivity to electrophilic compounds -Overexpression of ETGE-containing proteins can further activate NRF2 activity in a KEAP1 mutant background
Major Lab Ben Major Erica Cloer Kathleen Mulvaney Dennis Goldfarb Priscila Siesser Matt Walker Feng Yan Alex Rabinowitz
Hayes Lab Neil Hayes Matt Wilkerson
TCGA Research Network
Ning Zheng (U. Washington)
Questions?